Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity

Background

Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.

Methodology/Principal Findings

Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe₃O₄ were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.

Conclusions/Significance

The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.     

Assessing the effect of varying sequence length on DNA barcoding of fungi

DNA barcoding shows enormous promise for the rapid identification of organisms at the species level. There has been much recent debate, however, about the need for longer barcode sequences, especially when these sequences are used to construct molecular phylogenies. Here, we have analysed a set of fungal mitochondrial sequences - of various lengths - and we have monitored the effect of reducing sequence length on the utility of the data for both species identification and phylogenetic reconstruction. Our results demonstrate that reducing sequence length has a profound effect on the accuracy of resulting phylogenetic trees, but surprisingly short sequences still yield accurate species identifications. We conclude that the standard short barcode sequences ( approximately 600 bp) are not suitable for inferring accurate phylogenetic relationships, but they are sufficient for species identification among the fungi.     

Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro

We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei which contains C14 acyl and alkyl chains shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.     

Phenylalkyl backbone modified oligodeoxynucleotides, their synthesis and the influence of the alkyl chain length

Phenylalkyl modified phosphoramidites (alkyl chain length n = 1, 2, 3, 5; Fig. 1) were synthesised and incorporated into a DNA hexamer (5'-d(GCCp-GCG); p = place of modification). The obtained diastereomeres were separated by RP-HPLC. After hybridisation with the complementary DNA strand Tm-value and thermodynamic data were measured. The stability of duplexes depends on the linker length and the absolute configuration of the backbone modified oligodeoxynucleotides (Rp, Sp).     

The role of alkyl chain length in the inhibitory effect n-alkyl xanthates on mushroom tyrosinase activities

Sodium salts of four n-alkyl xanthate compounds, C2H5OCS2Na (I), C3H7OCS2Na (II), C4H9OCS2Na (III), and C6H13OCS2Na (IV) were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) in 10 mM sodium phosphate buffer, pH 6.8, at 293 K using UV spectrophotometry. 4-[(4-Methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed, competitive or uncompetitive inhibition for the four xanthates. For the cresolase activity, I and II showed uncompetitive inhibition but III and IV showed competitive inhibition pattern. For the catecholase activity, I and II showed mixed inhibition but III and IV showed competitive inhibition. The synthesized compounds can be classified as potent inhibitors of MT due to their Ki values of 13.8, 11, 8 and 5 microM for the cresolase activity, and 1.4, 5, 13 and 25 microM for the catecholase activity for I, II, III and IV, respectively. For the catecholase activity both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail of these compounds. The length of the hydrophobic tail of the xanthates has a stronger effect on the Ki values for catecholase inhibition than for cresolase inhibition. Increasing the length of the hydrophobic tail leads to a decrease of the Ki values for cresolase inhibition and an increase of the Ki values for catecholase inhibition.     

Study of thermodynamic properties of imidazolium-based ionic liquids and investigation of the alkyl chain length effect by molecular dynamics simulation

In this paper, structural and dynamical properties of five imidazolium-based ionic liquids (ILs) [amim]Br (a = methyl, ethyl, butyl, pentyl, hexyl) were studied by molecular dynamics simulations. United atom force field (UAFF) has been used for the representation of the interaction between ions. Good agreement with experimental data was obtained for the simulated density based on the UAFF. The calculated densities gradually decrease with an increase in the length of alkyl side chain, which is a result of weakening the electrostatic interaction between ions. The simulated heats of vaporisation are higher than that of non-ILs and decrease with an increase in temperature. Radial distribution function (RDF) was employed to analyse the local structure of ILs. Cation–anion RDFs show that the anions are well organised around the cation in two shells (0.41 and 0.6 nm). The velocity autocorrelation functions of the anion and cations show that the relaxation time increased with an increase in the length of the alkyl side chain. The diffusion coefficients of ions were calculated by mean square displacement of the centre of mass of the ions at 400 K. The calculated diffusion coefficients using UAFF agree well with other all atom force fields. Also diffusion coefficients decrease with an increase in the length of the alkyl side chain. The calculated transference numbers show that the cation contributes more than anion in the electrical current. The diffusion coefficients increase with temperature.     

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of N(ε)- and N(α)-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.     

Benzofuran-isatin hybrids tethered via different length alkyl linkers and their in vitro anti-mycobacterial activities

A series of novel benzofuran-isatin hybrids 6a–m tethered through different length alkyl linkers propylene, butylene, pentylene and hexylene were designed, synthesized and evaluated for their in vitro anti-mycobacterial activities against both drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (MTB) and cytotoxicity towards VERO cells. All hybrids with acceptable cytotoxicity in VERO cells (CC₅₀: 64 to >1024 μg/mL) also exhibited considerable anti-mycobacterial activities against both drug-susceptible and MDR-MTB strains with MIC in a range of 0.125–4 μg/mL. The SAR indicated that the length of the linker played a pivotal role on the activity, and the longer linker could enhance the activity. The most active hybrid 6d (MIC: 0.125 and 0.125 μg/mL) was comparable to or better than rifampicin (MIC: 0.5 μg/mL) and isoniazid (MIC: 0.06 μg/mL) against MTB H₃₇Rv, and was ≥256 folds more potent than rifampicin (MIC: 64 μg/mL) and isoniazid (MIC: >128 μg/mL) against MDR-MTB strain, but was less active than TAM16 (MIC: <0.06 μg/mL against the tested two strains). The hybrid 6d also showed low cytotoxicity towards VERO cell (CC₅₀: 128 μg/mL), but it was inferior to TAM16 in metabolic stability and in vivo pharmacokinetic profiles.     

Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide

The effects of phospholipid or detergent chain length on the structure and translational diffusion coefficient of the membrane-targeting peptide corresponding to the N-terminal amphipathic sequence of Escherichia coli enzyme IIA(Glc) were investigated by nuclear magnetic resonance (NMR) spectroscopy. Three anionic phospholipids (dihexanoyl phosphatidylglycerol, dioctanoyl phosphatidylglycerol, and didecanoyl phosphatidylglycerol) and four lipid-mimicking anionic detergents (sodium hexanesulfonate, 2,2-dimethyl-silapentane-5-sulfonate, sodium nonanesulfonate, and sodium dodecylsulfate) were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of those single-chain detergents on the helix length is more pronounced. The diffusion coefficients of the peptide/micelle complexes were found to correlate with the chain lengths of both the lipid and the detergent groups. Taken together, short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides such as cationic antimicrobial peptides. DSS does not form micelles by itself according to the diffusion coefficient data, but it does associate with this cationic peptide. Consequently, both DSS and its analog may be chosen as NMR chemical shift reference compounds depending on the nature of the biomolecules under investigation.     

Enhanced anti-influenza A virus activity of (-)-epigallocatechin-3-O-gallate fatty acid monoester derivatives: effect of alkyl chain length

A series of fatty acid monoester derivatives of (−)-epigallocatechin-3-O-gallate (EGCG) were prepared by one-pot lipase-catalyzed transesterification. The introduction of long alkyl chains enhanced anti-influenza A/PR8/34 (H1N1) virus activity 24-fold relative to native EGCG.     

Effects of alkyl chain length of gallate on self-association and membrane binding

Alkyl gallates are anticipated for their use as anti-bacterial and anti-viral agents. Although their pharmacological activities depend on their alkyl chain length, no mechanism has yet been clarified. As described herein, we investigated the membrane binding properties of a series of alkyl gallates using fluorescence measurement to elucidate their different pharmacological activities. Membrane binding of the alkyl gallates increased concomitantly with increasing alkyl chain length, except for cetyl gallate and stearyl gallate. Dynamic light scattering revealed that alkyl gallates with a long alkyl chain are prone to self-association in the solution. Membrane binding abilities of the alkyl gallates are correlated with anti-bacterial and anti-virus activities, as described in previous reports. The partition constants of the alkyl gallates to lipid membranes depend on the membrane components and the membrane phase. Self-association and lipid binding of the alkyl gallates might be primary biophysical factors associated with their pharmacological activities.     

Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

Immobilization of flavoproteins on silicon: effect of cross-linker chain length on enzyme activity.

The effect of cross-linker chain length on the activities of choline oxidase (ChO) and glucose oxidase (GOx) immobilized on oxidized silicon wafers has been investigated for the cross-linkers N-succinimidyl 4-maleimido-butyrate (GMBS) and N-succinimidyl 6-maleimidocaproate (EMCS). Enzyme activities were determined with an indirect fluorometric assay based on the production of hydrogen peroxide. Immobilization of ChO or GOx onto oxidized silicon with either cross-linker resulted in an 86-99% loss in enzymatic activity relative to the soluble form of the flavoprotein. However, the different cross-linkers had distinctly different effects on enzyme activity: EMCS-immobilized GOx was four times more active than GMBS-immobilized GOx; EMCS-immobilized ChO had a sevenfold higher activity than GMBS-immobilized ChO.     

Effect of ionic liquids alkyl chain length on horseradish peroxidase thermal inactivation kinetics and activity recovery after inactivation

The effects of different alkyl chain lengths of ionic liquids 1-ethyl-, 1-butyl- and 1-hexyl-3-methylimidazolium chloride, on the catalytic activity, thermal stability and deactivation kinetics of horseradish peroxidase were studied in the temperature range of 45–85 °C. The presence of 1-ethyl- and 1-butyl-ionic liquids up to 25 % (w/v) did not affect significantly the enzyme activity at 25 °C, whereas the addition of 1-hexyl-solvent resulted in lower activity of enzyme. Typical biphasic deactivation profiles were obtained and adequately fitted by a bi-exponential equation. When increasing ionic liquids concentration up to 25 % (w/v), the second phase of deactivation became more prominent, till leading to apparent first-order kinetics. Occurrence of activity regain, following thermal deactivation was found, reaching up 60–80 % of the initial activity, especially in 1-hexyl-3-methylimidazolium chloride. Activity regain was particularly noticeable in the first phase of deactivation. Temperature sensitivity of the Soret band maxima indicated that the enzyme prepared in buffer or 1-hexyl-3-methylimidazolium chloride had similar conformational changes in the haem region, but no correlations were found with activity decrease.     

Stochastic properties of motoneuron activity and the effect of muscular length

The activity of single motor units contributing to small tonic isometric contractions in human muscle at different muscular lengths was analyzed. The form of motor unit firing patterns shows that the interspike intervals compose independent sequences with about a 10% coefficient of variation and have a gamma distribution. The variability and the distribution shape curves show that as the mean interval decreases the variance also decreases and the interval density function becomes more symmetric. More significant is the fact that the form of the firing pattern remains unchanged when a motor unit has the same mean interval but with the muscle at different lengths. Comparison of these facts with experimental data from neuron models and cat motoneurons indicates that in the human the only relevant input-output relationship in motoneurons is that the net excitation adjusts the firing rate.     

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.     

The effect of the chain length of polynucleotides on their binding with platinum complexes.

The effect of the length of polynucleotides on their binding with platinum complexes was studied. The highest reaction rate was observed in the reaction with guanosine-containing polynucleotides, whereas cytidine- and adenosine-containing polynucleotides were much less efficient. The monoaqua-forms of the platinum complexes exhibited the highest reactivity in the interaction with polynucleotides in solution. The mechanism implies the formation of the monodentate complex at the first stage which is transformed into the corresponding bidentate complex of chelate type at the second stage. Increase in the length of the polynucleotide chain was shown to enhance its interaction with the platinum complexes.     

Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains. The DNA release from the amphiphile was tuned by altering the length and position of the ester linkage in the hydrophobic chains of the lipids through the preparation of five new amphiphiles. The amphiphiles and corresponding lipoplexes were characterized by DSC, TEM, and X-ray, as well as evaluated for DNA binding and DNA transfection. For one specific charge-reversal lipid, stable lipoplexes of approximately 550 nm were formed, and with this amphiphile, effective in vitro DNA transfection activities was observed.